1. Field of the Invention
The present invention relates generally to sensors for specific nucleic acid sequences. More particularly, the present invention relates to a real-time sensor system for the detection of target live agent deoxyribonucleic acid, such as would be useful in the detection of biological warfare agents.
2. Description of Related Art
The detection of bacteria and viruses that pose a threat to human populations is an invaluable capability. With early detection of a harmful microorganism, persons located in the vicinity of an infected area may be notified so that they might take necessary precautions for their protection, such as fleeing to a safe haven, consuming appropriate antibiotics, or donning protective gear.
While the proliferation of harmful air-borne or water-borne microorganisms in an area might be a natural occurrence or, at least, an unintended result of human interference (e.g., the contamination of bodies of water by raw sewage), the potential for rogue governments and terrorists to employ biological warfare agents (BWA's) against troops and civilian populations is an increasing concern that makes the need for sensor systems for harmful microorganisms all the more urgent. The sheer variety of BWA's that might be employed requires that a sensor system should be highly sensitive and highly selective with regard to target live agents. Examples of potential BWA's include anthrax, typhoid fever, smallpox, and valley fever. Given the speed with which BWA's might adversely affect a population, it would be highly desirable that the sensor system offer real-time detection.
There are available methods by which harmful microorganisms might be detected, such as antibody-based techniques and gene-probe assays. However, antibody-based techniques suffer from poor specificity and are not adaptable to field applications. Gene-probe assays have been considered a better alternative to antibody-based techniques alone, but gene-probe assays have been known to require numerous time-consuming steps which are difficult to automate. Neither antibody-based techniques nor traditional gene-probe assays offer the real-time sensor capabilities and the specificities needed to detect harmful microorganisms such as BWA's.
Recently, techniques have been developed which enable the highly specific and sensitive detection of live agents by addressing the genetic material of the biological warfare agent itself. These techniques are disclosed in (1) a patent application having International Publication Number WO 95/15971 (entitled "Nucleic Acid Mediated Electron Transfer"), naming Thomas J. Meade and Jon F. Kayyem as inventors and having been assigned on its face to California Institute of Technology; and (2) a publication entitled "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors" (Angew. Chem. Int. Ed. Engl., Vol. 34, No. 3, pp. 352-354 (1995)), written by Thomas J. Meade and Jon F. Kayyem.
The method developed by Meade and Kayyem (hereinafter Meade et al) provides for the site-selective modification of nucleic acids with redox active moieties such as transition metal complexes. Specifically, Meade et al demonstrate the placement of ruthenium-containing electron donor and electron acceptor groups onto the ribose backbone of a single strand of DNA, allowing the placement of a ruthenium atom at or near each end of a single strand of DNA. The significance of this feat lies in the electrical conductance differential between ruthenium-doped single strands of DNA compared to that of a double helix comprising one such single strand and a non-doped complementary strand. In general, a double strand of polynucleotide is about one million times more electrically conductive than a single strand. When a ruthenium-doped single strand is combined with its non-doped complementary strand, one of the ruthenium-containing groups serves as an electron source and the other as an electron sink, such that electrons flow back and forth between these two groups. Thus, the double helix creates a highly conductive path along its molecular axis between the electron donor and the electron acceptor that does not exist in a single strand of DNA. In addition to serving as electrical connectors into and out of the molecule, ruthenium atoms have the added virtue of neither disrupting nor distorting the overall shape of the DNA backbone.
Prior to the method of Meade et al, the long range transference of electrons in a DNA matrix was hindered by such factors as (1) the random distribution and mobility of the electron donor and the electron acceptor pairs; (2) the potential short distances between the donor and acceptor; and (3) the loose and reversible association of donors and acceptors. Meade et al overcome these obstacles by teaching a method directed to the modification of nucleic acids with electron transfer moieties covalently attached to specific sites of nucleic acids on a single strand of DNA in a way that creates no stearic hindrance to hybridization to form a duplexed strand pair. The resulting doped DNA strand is capable of hybridizing to a complementary target sequence in a single stranded nucleic acid and thereafter rapidly transferring electrons between the donor and acceptor.
The method of Meade et al specifically involves the synthesis of two sets of complementary oligonucleotide strands modified first with a terminal aminoribose and then covalently modified with electron donor and acceptor moieties, such as redoxactive ruthenium complexes. Since the ruthenium complexes will react with the heterocyclic nitrogen atoms of the bases, the bases are protected by employing an unmodified complementary strand as a large hydrogen-bonded blocking group. After reaction with the ruthenium complex, the complementary strand is removed and the modified oligonucleotide is purified in accordance with known procedures.
By employing the methods of Meade et al, one may discriminate between DNA strands that are identical to the original and those that differ by, for example, one base pair out of fifteen pairs (i.e. a match of 14 of the 15 base pairs in a strand), or by more than one base pair, by observing the electrical conductivity of an oligonucleotide. Meade et al have thus developed an electrical method of distinguishing between different sequences of DNA. Meade et al expressly contemplate employing their method of DNA strand detection in the creation of bioconductors and diagnostic probes, such as for medical applications.
A need remains for a real-time sensor system for harmful microorganisms that offers highly sensitive and highly selective characteristics; that may be employed for environmental sampling in the field, such as air and water sampling; and that is capable of alerting both those in its immediate vicinity as well as those at remote locations of the presence and type of microorganisms detected. Further, the sensor should be conveniently sized for use in the field and should be relatively inexpensive to produce and operate.